mecrl-meci, and fema-femb in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus

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1 ANTIMICROBiAL AGENTS AND CHEMOTHERAPY, Dec. 1992, p /92/ $2./ Copyright X 1992, American Society for Microbiology Vol. 36, No. 12 Survey of the Methicillin Resistance-Associated Genes meca, mecrl-meci, and fema-femb in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus ROCIO L. HURLIMANN-DALEL, CRISTINA RYFFEL, FRITZ H. KAYSER, AND BRIGITTE BERGER-BACHI* Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Received 27 March 1992/Accepted 14 September 1992 The restriction site polymorphism of the chromosomal femab region and the first appearance of the regulatory element mecri-meci associated with the methicillin resistance determinant (mec) were analyzed in 192 initially methicillin resistant (Mc') Staphylococcus aureus clinical isolates collected between 1965 and 199O in the Zurich area. Forty-three of the strains lost the resistance spontaneously. All isolates that were still Mcr hybridized with meca, the gene for the low-affinity penicillin-binding protein PBP 2'. Mcr strains isolated before 1977 lacked sequences that hybridized with mecri-meci, a regulatory element controlling the expression of meca; exceptions to this were one strain isolated in 1966 and one strain isolated in The size of the EcoRV fragment carryingfema, a chromosomally encoded factor involved in pentaglycine side chain formation of the peptidoglycan and essential for the expression of methicillin resistance, was conserved in all strains but one, which was susceptible to methicillin even though it carried a functional meca gene. The methicillin susceptibility of this particular strain was presumably due to a spontaneous fem4-like mutation. The 192 strains belonged to seven different EcoRV restriction fragment patterns recognizable with a 1.5-kb probe covering thefemab region. Some 93% of the 149 Mcr strains belonged to pattern A, and the remaining Mcr strains shared patterns A' and B. The 42 isolates which spontaneously lost their resistance upon storage and revival represented all seven different patterns. This strong conservation offema suggests an important role for fema4 in cell wall metabolism and methicillin resistance. Methicillin-resistant (Mcr) staphylococci were described soon after the introduction of methicillin, the first penicillinase-resistant P-lactam antibiotic, into clinical use (11). The similar antibiotic resistance and phage typing pattern of the early strains suggested that they spread initially from a single clone of Mcr Staphylococcus aureus (14). In an epidemiological analysis of natural populations of S. aureus by multilocus enzyme electrophoresis, Musser and Selander (19) showed that 78% of Mcr isolates belong to one clone and that the remaining strains are clustered in a few electromorph clones. In the time that has elapsed since the first use of methicillin, old clones might have been displaced by new ones of different origin or with genetically different methicillin resistance determinants (mec). Clones of Mcr staphylococci which do not involve the mec determinant have appeared (1, 25, 28). The genes analyzed in the present study were (i) meca, which encodes the low-affinity penicillin-binding protein PBP 2', the main factor responsible for the methicillin resistance (1, 27, 29), and (ii) a regulatory element encoding mecrl-meci, which is associated in some strains with meca. This element, termed mecr in an earlier report (26), consists of two open reading frames coding for a putative transmembrane-signaling protein MecRl and a putative repressor MecI (9, 22, 23). The presence of this regulatory element strongly represses meca transcription in the absence of an inducer (21). Repression in some strains is very strong, and methicillin resistance is induced very slowly, so that it was postulated that these strains might be misinterpreted to be susceptible strains in standard disk diffusion tests (23). We * Corresponding author determined the first appearance of this regulatory element in our collection of Mcr S. aureus strains. Additional genes that were analyzed werefema andfemb, which map close to trpa on the chromosome and thus are not linked to the mec resistance determinant (6, 7). These two factors make up part of the chromosomal genes that occur naturally in susceptible as well as resistant S. aureus strains. It is known that, in addition to mec, these additional factors are essential for the expression of methicillin resistance (4, 7, 8, 13, 18). Insertional inactivation of fema or femb by TnS51 increases the susceptibility of methicillinsusceptible (Mcs) and Mcr S. aureus strains to P-lactams (6, 8). FemA has been characterized, and its natural task has been deduced to be some step in pentaglycine side chain formation, which is characteristic of the highly cross-linked peptidoglycan in S. aureus (15). We confirmed in the present study that these two genes were present in all Mcr strains analyzed; in the analyzed strains, the size of the restriction fragment carryingfema was highly conserved. MATERMILS AND METHODS Bacterial isolates and susceptibility tests. Mcr S. aureus strains isolated during the first 3 months of the year at the Institute of Medical Microbiology in Zurich were collected and stored as lyophilized cultures. The collection started in The clinical isolates were from patients of the University Hospital of Zurich and other hospitals in the Zurich area. The criteria used to classify them as Mcr when they were first collected was growth of undiluted overnight broth cultures that were spot inoculated onto agar plates containing various concentrations (6.25 to 5 mg/liter) of methicillin and 5% NaCl. The cultures were incubated at 3 C for 48 h.

2 2618 HURLIMANN-DALEL ET AL. Strains that grew at concentrations of 12.5 mg/liter or greater were termed resistant. From this collection a maximum of 1 Mcr S. aureus strains were chosen randomly to represent each year in the study. Not more than one strain per patient was incorporated into the study. The last few years were represented by only five or fewer isolates because less than 1% of S. aureus strains from the University Hospital were Mcr. After revival of the strains, their identities were checked by standard methods (12). Methicillin resistance was determined by disk diffusion tests according to the standard of the National Committee for Clinical Laboratory Standards (2). For some strains, the MIC of oxacillin was determined with E-strips (2). Strains that spontaneously lost their methicillin resistance upon revival were also incorporated into the study. As controls we included Mcr S. aureus. BB27, a derivative of NCTC 8325 carrying a mec determinant of the meca type; its susceptible parent strain BB255; and Mcr Staphylococcus epidermidis WT55 (of the meca4- mecri-meci type) (23). Molecular biological tchniques. Chromosomal DNA was isolated by standard procedures by using lysostaphin to lyse the cell walls (16). Digests with different restriction endonucleases were performed according to the recommendations of the manufacturer (Boehringer, Mannheim, Federal Republic of Germany). Separation of the DNA digests by agarose gel electrophoresis, transfer to membranes, hybridization with radioactively labeled DNA probes, and exposure of the membranes to X-ray ifims were done as described previously (16). DNA probes. The probes used in the present study are listed in Fig. 1. The meca probe was the 1-kb XbaI-BglII fragment covering meca4 (24). The probe for the regulatory region was the 2.2-kb HidIII fragment covering part of mecri and meci cloned from strain WT55 (22, 23). The probe forfema was a 2.2-kb EcoRV fragment, and forfemb it was a 1.1-kb EcoRV fragment. Restriction fragment polymorphism in thefemab region was probed with the 1.5-kb PstI fragment (6). Trans tion. Transformation of strain BB255 with total chromosomal DNA from clinical isolates was performed by the CaC12 method described earlier (7). Selection of transformants was done on 5 mg of methicillin per liter. RESULTS Epidemiology of the Mcr S. awrus isolates. The statistics of the Institute of Medical Microbiology in Zurich indicate that during the time period from 1965 to 199 two outbreaks of Mcr S. aureus occurred. In 1969 over 27% of the S. aureus isolates were resistant to methicillin, and in 1981 there was a peak with 15% Mcr strains (Fig. 2). In recent years fewer than 1% of S. aureus strains showed Mcr, so that only a few Mc' isolates are represented in the last years covered by our study. Of a total of 192 strains analyzed, 42 spontaneously lost their methicillin resistance upon storage and revival; no resistant clones could be isolated from cultures of those strains. Since those strains were initially tested very carefully, in most cases by population analysis resulting in the typical heterogeneous methicillin resistance phenotype, before being incorporated into the collection, and since we previously observed the spontaneous loss of the mec determinant by storage of strains at room temperature (3), the strains were also incorporated into our study to test whether they belonged to a specific clone. The frequency of loss of the mec determinant was found to be unevenly distributed A meca probe S. an BB27 S. apidwmidi WT55 mecrl-meci probe B 1 kb 1 kb $ $ = = = -~ A- ~ = = moca = meci mecrl moca '1 U U 2Z temab probe fema probe 19mB probe FIG. 1. Restriction maps. (A) mec determinant of strain BB27, which lacks the regulatory region, and the mec determinant of strain WT55, which contains the regulatory region. The dotted line in the map of strain BB27 shows where the homology between both mec determinants ends. Open reading frames and the direction of transcription are indicated by arrows. meca encodes the low-affinity penicillin-binding protein PBP 2', and mecri and meci (27) are open reading frames encoding a putative tranmembrane P-lactam-sensing protein and a repressor, respectively (21); both are encoded by the regulatory region of meca (23). The probes used in Southern hybridizations are shown by heavy bars. (B) femab region of the chromosome of strain BB27. The open reading fames fema and femb are indicated. The EcoRV fragments that hybridized with the femab probe are numbered consecutively by decreasing size; the corresponding fragments in strain BB27 are indicated in the control lane (lane BB27) of Fig. 4. over the years; except for the isolates obtained in 1967, the older isolates seemed more stable (Fig. 3). Correlation of Mc' with meca. HindlI digests of all 192 strains were probed with meca, the structural gene for PBP 2'. In all strains, methicillin resistance correlated with the presence of a 4- to 4.2-kb HindIII fragment labeled with meca. The only exception was one strain isolated in The MIC of oxacillin was.1 mg/liter for this particular strain; it lost its methicillin resistance, although it hybridized ; I 1 on ANTBOCROB. AGENTS CHEMOTHER. l year Epidemiology of McJ and Mce strains as the percentage FIG. 2. of Mcr S. aureus strains isolated from hospitalized patients at the Institute of Medical Microbiology in Zurich.

3 VOL. 36, 1992 EPIDEMIOLOGY AND femab PATITERNS IN S. AUREUS 2619 c 1- c,' m m11 A A' B C D E F *i Lo E 1 _ year FIG. 3. Number of S. aureus clinical isolates from each year analyzed in the present study. a, Mcr strains carrying meca only without the regulatory region; *, Mcr strains carrying the mec determinant with the regulatory region; A, Mcs strains which did not hybridize with the meca gene, which are plotted as negative values. with meca. Transformation of the susceptible strain S. aureus BB255 with DNA isolated from the strain isolated in 1985 yielded Mcr colonies, showing that the mec determinant was functional. Susceptibility must therefore be a consequence of a chromosomal mutation(s), presumably in the fema region, since this strain was shown to have an unusual EcoRV restriction pattern in the fema region, as discussed below. Occurrence of the regulatory element mecri-meci. For our collection of strains, the agar disk diffusion test with the 1-,ug oxacillin disk allowed us to recognize the Mcr strains carrying meca as well as those carrying meca with the regulatory region mecrl-mecl. On the one hand, some strains had rather large inhibition zones with a few single colonies growing within the zone; these were presumably low-level, heterogeneously Mcr strains with only a few higher-level resistant subclones. This behavior was apparent in meca as well as in meca-mecri-meci type strains. On the other hand, presumably high-level, homogeneously resistant strains that grew up to the oxacillin disk were found in equal numbers among the strains that carried either type of mec determinant. The regulatory element with the genes mecrlmeci appeared in a Mcr strain first in 1966 and then in It was only from 1977 on that larger numbers of these mecrl-meci-containing strains were found. By 1984, Mcr strains of the meca-mecrl-meci type had displaced all other Mcr strains. Mcr strains with the regulatory region disappeared in 1987 (Fig. 3). Mcr S. aureus strains of the meca type were more common in our collection (117 strains) than those of the meca-mecrl-meci type (33 strains). The first outbreak of Mcr strains in 1969 was caused by strains carrying meca only. Strains of the meca-mecrl-meci type were responsible for the second outbreak of Mcr S. aureus in This epidemic was due to a strain introduced into the University Hospital by a patient with multiple injuries from Libya (17). mecri-meci was always associated with the presence of meca in our strain collection. Presence offema andfemb. The strains were screened for the presence of fema, the chromosomal gene essential for the expression of resistance. The 2.2-kb size of the EcoRV fragment 4 carryingfema was conserved in all Mcr strains as well as in the Mcs strains, which had lost the resistance. The only exception was the one strain isolated in 1985 (see 3- fema 4 5- femb 6- FIG. 4. EcoRV restriction fragment patterns in the femab region. Lane A, Southern blot of EcoRV digests of chromosomal DNA isolated from selected clinical isolates probed with the 1.5-kb PstI fragment covering the femab region. The control lane (lane BB27) shows Mcr strain BB27 with pattern A. Its EcoRV fragments are numbered according to their sizes; their corresponding map positions are shown in Fig. 1. The pattern types are indicated above the lanes. Differential hybridizations with the fema and the femb probes were done earlier in order to localize the fragments that hybridized with fema and femb. Their positions on the gel are indicated. The fused band that hybridized to fema is indicated by a dot; those that hybridized to femb are indicated by arrowheads. above), which, even though it carried meca, had a Mcs phenotype. The 2.2-kb EcoRV fragment 4 was substituted in this particular strain by a band of approximately 3.8 kb, indicated by the dot in Fig. 4, lane E. This strain presumably had a deletion or mutation which extended over the EcoRV site upstream offema and fused the fema fragment 4 to the adjacent upstream fragment 1 (Fig. 1B). Hybridizations with the 1.2-kb EcoRVfemB gene carrying fragment 6 showed the presence of this fragment in 184 strains, represented by the patterns A, A', C, D, and E in Fig. 4. In six strains the band that hybridized to femb was larger than fragment 6 and is shown by arrows in Fig. 4, lanes B and F. Restriction site polymorphism in thefemab region. EcoRV digests were probed with the 1.5-kb PstI fragment covering the entire femab region and adjacent sequences; six fragments in the control strain BB27 were labeled, as shown in Fig. 1 and 4. The clinical isolates yielded seven different restriction patterns (Fig. 4 and Table 1). Pattern A, the same as the pattern in strain BB27, was found in 94% of all Mcr strains analyzed. In 1986 a new pattern, pattern A', appeared in Mcr strains. Pattern A' was similar to pattern A except for the size of the two largest fragments (Fig. 4). Two Mcr strains were of the B pattern. These three patterns (patterns A, A', and B) were found in all Mcr strains, independently of the genetic type of the mec determinant. The Mcs strains in our collection belonged to the same patterns (A, A', and B) and to the additional patterns C, D, E, and F. Noteworthy is that over one-third of the strains which lost the resistance were of pattern D. Pattern C was

4 262 HURLIMANN-DALEL ETAL.ANTimicRoB. AGENTs CHEMOTHER. TABLE 1. Distributions of the different S. aureus isolates among EcoRV patterns in the femab region No. of strains Year of Mce (meca or isolation meca-mecri- Mc' (no mecap' McS (meca), me pattern EC A A' B A A' B C D F S Total Mc? a strains containing the mec determinant with and without the regulatory b region. Mce strains whidh lost resistance after revival and which did not hybridize with meca or with the regulatory region. Cfen.A M? mutant strain carrying meca. represented by only three strains. There major rearrangements had taken place, but the rearrangements apparently involved neither fragment 4 (fema) nor fragment 6 (femb). Pattern E was represented by the one Mca strain discussed above; the strain carried a functional meca gene. In that strain, fragment 4 seemed to be fused to fragment 1 presumably by a rearrangement or mutation encompassing the upstream EcoRV site, leading to a fena-iike phenotype. Pattern F was found only in one susceptible strain which probably had a deletion that fused fragment 6 (frnlb) with fragment 3, since the size of the fusion product was smaller than the combined size of the missing fragments 6 and 3. DISCUSSION An intriguing finding of the present study was that 22% of Mer strains had lost their resistance after lyophilization, storage, and revival. The loss of resistance was unevenly distributed over the years, but instability was more often found in recent multiply Mcr S. aureus strains, suggesting that loss of the mec determinant is strain dependent. We could not reconstruct at which stage of handling the inethicillin resistance was lost. Presumably, the timfe that elapsed between testing and final lyophilization might have preferentially allowed the susceptible mutants of unstable strains to survive and grow. Cycles of storage and subculturing might have favored deletion formation. We repeatedly observed the spontaneous loss of Mcr, especially in clinical S. epidermidis isolates (unpublished data), and documented the loss of the mec-specific sequences (3) in a clinical isolate of Mcr S. aureus after storage of the culture in agar slants at room temperature. Another hypothesis might be that these initially Mcr strains might never have carried a mec determinant but were Mcr by mechanisms other than meca, for instance, by altered PBPs with a lower affinity to 1-lactams, and that this trait reverted readily. This latter hypothesis is unlikely, since in all of the Mcr clinical isolates in our area, methicillin resistance is always found to be associated with the presence of mec4. It is possible, on the one hand, that a specific genetic background, as represented by those strains withfemrab pattern D, increased the instability. On the. other hand, deletion of mec might have occasionally caused rearrangements elsewhere in the chromosoine, leading to patterns D, C, or F, which were not represented in the resistant strains. Analysis of initially susceptible S. aureus strains will tell us whether these femab EcoRV restriction patterns belong to clonal lineages specific for Mcr strains. It was interesting to find an apparently naturalfem mutant among the spontaneously occurring Mc8 strains. Its mec determinant was functioning, as was proven by transformation into a susceptible strain, which yielded resistant transformants. Consequently,' the inability to express resistance to methicillin is most likely due to inactivation of one of the fem factors, presumablyferna orfemb, since it was the only strain with a rearrangement that removed the EcoRV site close to the origin of transcription of the femab operon. In all other strains, the 2.2-kb EcoRV fragment carrying fera was exceptionally well conserved, supporting the fact that FemA and probably FemB as well play an important role in cell wall metabolism. There was a clear preference in Mcr strains for specific strains with pattem A in the femnab region. A new strain with the A' pattern has apparently displaced the A-pattern strains in Ziirich. Whether the clonal hypothesis in Mcr staphylococci holds true for the entire genome or just for this restricted chromosomal region will be answered only after an appropriate number of initially susceptible staphylococci are compared by the same method. Since all Mcr strains hybridized with meca, we conclude that in the Zurich area the mec determinant is the major -reason for methicillin resistance in S. aureus and that Mcr strains with altered penicillin-binding proteins still play a minor role in our medical institutions. Since we did not find any correlation between phenotypic expression of resistance in strains with or strains without the regulatory region, we suggest that heterogeneity and slow induction of methicillin resistance is dependent on additional unknown strain-specific factors. Strains that lack the regulatory element, in comparison with those that carry mecrimeci, have a high level of sequence homology through the meca gene close to the HindIII site upstream of meca, at which point the sequence homology changes abruptly (Fig. 1) (5). In the former strains, which have a constitutive meca, synthesis of the mecri open reading frame is truncated and ends at an accumulation of stop codons (21, 22). One hypothesis is that strains of the meca type were derived from those of the meca-mecri-mecl type by deletion of the regulatory region and would therefore have appeared later. Unexpectedly, in our collection of Me strains, those without the regulatory region appeared before strains with this region. Since from the time that we started our collection in 1965 all possible precautions were taken so that we would be

5 VOL. 36, 1992 EPIDEMIOLOGY AND femab PATlFERNS IN S. AUREUS 2621 able to recognize and enhance expression of methicillin resistance in clinical isolates, we are confident that the early strains, even if of very low resistance, would have been identified as Mcr and collected. Results from our strain collection suggest that two main epidemic strains with genetically different mec determinants were present in the time period covered by the study. It is known that the resistance level is not dependent on the mec determinant alone or on the amount of PBP 2', but is likely a feature of chromosomally encoded genes of the S. aureus genome. Although the femab region, which is involved in peptidoglycan biosynthesis, is essential for methicillin resistance expression, it does not seem to be responsible for homogeneous or heterogeneous methicillin resistance (8). The genetic backgrounds of some Mcr strains probably differ significantly from each other, even if they share the same or similar pattern in the feiiab region. The surprisingly well-conserved size of the 2.2-kb EcoRV fragment which carries fema suggests that FemA plays an important role in cell metabolism and corroborates its importance in methicillin resistance. It will be an interesting challenge to identify the unknown chromosomal genes which discriminate between homogeneous and heterogeneous methicillin resistance. ACKNOWLEDGMENT This work was supported by Swiss National Science Foundation grant REFERENCES 1. Archer, G. L., and E. Pennell Detection of methicillin resistance in staphylococci by using a DNA probe. Antimicrob. Agents Chemother. 34: Baker, C. N., S. A. Stocker, D. H. Culver, and C. Thornsberry Comparison of the E-test to agar dilution, broth microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria. J. Clin. 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